Mutant reverse transcriptase

ABSTRACT

A mutant MMLV reverse transcriptase that may have an improvement in one or more properties is provided. For example, the present reverse transcriptase is believed to be more efficient relative to other commercially available MMLV reverse transcriptase variants, particularly for templates with a higher GC content.

BACKGROUND

Reverse transcriptases are multi-functional enzymes with three enzymaticactivities including RNA- and DNA-dependent DNA polymerization activity,and an RNaseH activity that catalyzes the cleavage of RNA in RNA-DNAhybrids. Mutants of reverse transcriptases have disabled the RNaseHmoiety to prevent unintended damage to the mRNA. These enzymes thatsynthesize complementary DNA (cDNA) using mRNA as a template were firstidentified in RNA viruses. Subsequently, reverse transcriptases wereisolated and purified directly from virus particles, cells or tissues.(e.g., see Kacian et al., 1971, Biochim. Biophys. Acta 46: 365-83; Yanget al., 1972, Biochem. Biophys. Res. Comm. 47: 505-11; Gerard et al.,1975, J. Virol. 15: 785-97; Liu et al., 1977, Arch. Virol. 55 187-200;Kato et al., 1984, J. Virol. Methods 9: 325-39; Luke et al., 1990,Biochem. 29: 1764-69 and Le Grice et al., 1991, J. Virol. 65: 7004-07).More recently, mutants and fusion proteins have been created in thequest for improved properties such as thermostability, fidelity andactivity.

Copying RNA can be inhibited by the presence of RNA secondary structurewhich can stall cDNA synthesis resulting in truncated cDNA molecules.The formation of secondary structure can be avoided at highertemperature. While this also reduces non-specific priming and therebyincreases reverse transcriptase fidelity, length and yield of cDNA.However, RNA integrity can be compromised by lower enzyme activity atelevated temperatures. Further improvements are desirable to obtainoptimum performance of the enzymes in library synthesis and NextGensequencing.

SUMMARY

A mutant Moloney murine leukemia virus (MMLV) reverse transcriptase thatmay have an improvement in one or more properties is provided. Forexample, the present reverse transcriptase is believed to be moreefficient relative to other commercially available MMLV reversetranscriptase variants, particularly for templates with a higher GCcontent. In some embodiments, use of the present MMLV reversetranscriptase may increase the proportion of full length cDNA moleculesat a temperature that is higher than 42° C. (e.g., a temperature in therange of 45° C. to 60° C.). The present MMLV reverse transcriptase hasat least 7 amino acid substitutions relative to the wild type MMLVreverse transcriptase.

This disclosure provides, among other things, a polypeptide comprisingat least 300 contiguous amino acids of SEQ ID NO:1. The polypeptide maycomprise at least amino acid residues 24-335 of SEQ ID NO:1 and, in someembodiments may have a truncated N-terminus relative to SEQ ID NO:1. Insome embodiments, the polypeptide may comprise the entire contiguoussequence of SEQ ID NO:1.

In some embodiments, the polypeptide may additionally comprise an aminoacid sequence that is at least 90% identical to at least 286 contiguousamino acids of SEQ ID NO:2, where the additional amino acid sequence isC-terminal to the at least 300 contiguous amino acids of SEQ ID NO:1. Insome embodiments, the polypeptide may additionally comprises apurification tag and/or an exogenous sequence-specific DNA bindingdomain.

In some embodiments, the polypeptide may have reverse transcriptaseactivity. In these embodiments, the polypeptide may or may not have anRNAseH activity in addition to the reverse transcriptase activity.

In general, a method for reverse transcribing an RNA template is alsoprovided. In some aspects, the method may comprise: (a) combining aprimer, an RNA template and a reverse transcriptase comprising: i. atleast 300 contiguous amino acids of SEQ ID NO:1 and optionally ii. anamino acid sequence that is at least 90% identical to at least 286contiguous amino acids of SEQ ID NO:2 that is C-terminal to the at least300 contiguous amino acids of SEQ ID NO:1, to produce a reaction mix and(b) incubating the reaction mix to produce cDNA copied from the RNAtemplate.

In some aspects, the reaction mix may comprise a template switchingoligonucleotide and in other aspects, the reaction mix may incubated attemperature that is higher than 42° C., e.g., at a temperature in therange of 45° C. to 65° C. The primer in the reaction mix oligo-dTprimer, a random primer or a gene-specific primer, for example. As notedabove, in some cases, the polypeptide may comprise an exogenoussequence-specific DNA binding domain and, may or may not have RNAseHactivity.

In general, a method is provided for reverse transcribing an RNAtemplate wherein the population of cDNA molecules produced by the methodmay be at least 20%, at least 40%, at least 60%, or at least 80% fulllength. In other aspects, the method may comprise transcribing, withincreased efficiency compared with previously available reversetranscriptases, GC rich template molecules using embodiments of thereverse transcriptase described above where the template molecules mayhave at least 20%, 30%, 40%, 50%, 60%, 70% or 80% GC content. Inembodiments, the cDNA product of the GC rich template may be at least20%, at least 40%, at least 60%, or at least 80% full length.

These and other features of the present teachings are set forth herein.

BRIEF DESCRIPTION OF THE FIGURES

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way.

FIG. 1 shows some of the components used in the template switching assaydescribed in the Examples section.

FIG. 2 illustrates how reverse transcription efficiency can bequantified.

FIG. 3 is a bar chart showing the relative reverse transcriptionefficiencies of two commercially available MMLV reverse transcriptasevariants (SuperScript® IV (Life Technologies, Carlsbad, Calif.) andProtoScript® II (New England Biolabs, Ipswich, Mass.) and the M19Hvariant in copying RNA templates with differing GC content.

DETAILED DESCRIPTION OF EMBODIMENTS

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods and materials are described.

All patents and publications, including all sequences disclosed withinsuch patents and publications, referred to herein are expresslyincorporated by reference.

Numeric ranges are inclusive of the numbers defining the range. Unlessotherwise indicated, nucleic acids are written left to right in 5′ to 3′orientation; amino acid sequences are written left to right in amino tocarboxy orientation, respectively.

The headings provided herein are not limitations of the various aspectsor embodiments of the invention. Accordingly, the terms definedimmediately below are more fully defined by reference to thespecification as a whole.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton, et al., DICTIONARYOF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, NewYork (1994), and Hale & Markham, THE HARPER COLLINS DICTIONARY OFBIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with thegeneral meaning of many of the terms used herein. Still, certain termsare defined below for the sake of clarity and ease of reference.

As used herein, the term “reverse transcriptase” refers to any DNApolymerase that can copy first-strand cDNA from an RNA template. Suchenzymes are commonly referred to as RNA-directed DNA polymerases andhave IUBMB activity EC 2.7.7.49. In some cases, a reverse transcriptasecan copy a complementary DNA strand using either single-stranded RNA orDNA as a template. MMLV reverse transcriptase is the reversetranscriptase of the Moloney murine leukemia virus.

As used herein, the term “template” refers to the substrate RNA for thereverse transcriptase to make cDNA. A template may be complex (e.g.,total RNA, polyA+ RNA, mRNA, etc.) or not complex (e.g., an enriched RNAor an in vitro transcribed product).

The term “cDNA” refers to a strand of DNA copied from an RNA template.cDNA is complementary to the RNA template.

A “mutant” or “variant” protein may have one or more amino acidsubstitutions, deletions (including truncations) or additions (includingdeletions) relative to a wild-type. A variant may have less than 100%sequence identity to the amino acid sequence of a naturally occurringprotein but may have any amino acid that is at least 80%, at least 85%,at least 90%, at least 95%, at least 97%, at least 98% or at least 99%identical to the amino acid sequence of the naturally occurring protein.A fusion protein is a type of protein composed of a plurality ofpolypeptide components that are unjoined in their naturally occurringstate. Fusion proteins may be a combination of two, three or even fouror more different proteins. The term polypeptide includes fusionproteins, including, but not limited to, a fusion of two or moreheterologous amino acid sequences, a fusion of a polypeptide with: aheterologous targeting sequence, a linker, an immunologically tag, adetectable fusion partner, such as a fluorescent protein,β-galactosidase, luciferase, etc., and the like. A fusion protein mayhave one or more heterologous domains added to the N-terminus,C-terminus, and or the middle portion of the protein. If two parts of afusion protein are “heterologous”, they are not part of the same proteinin its natural state.

The term “non-naturally occurring” refers to a composition that does notexist in nature. Variant proteins are non-naturally occurring. In someembodiments, “non-naturally occurring” refers to a protein that has anamino acid sequence and/or a post-translational modification patternthat is different to the protein in its natural state. A non-naturallyoccurring protein may have one or more amino acid substitutions,deletions or insertions at the N-terminus, the C-terminus and/or betweenthe N- and C-termini of the protein. A “non-naturally occurring” proteinmay have an amino acid sequence that is different to a naturallyoccurring amino acid sequence (i.e., having less than 100% sequenceidentity to the amino acid sequence of a naturally occurring protein)but that that is at least 80%, at least 85%, at least 90%, at least 95%,at least 97%, at least 98% or at least 99% identical to the naturallyoccurring amino acid sequence. In certain cases, a non-naturallyoccurring protein may contain an N-terminal methionine or may lack oneor more post-translational modifications (e.g., glycosylation,phosphorylation, etc.) if it is produced by a different (e.g.,bacterial) cell.

In the context of a nucleic acid, the term “non-naturally occurring”refers to a nucleic acid that contains: a) a sequence of nucleotidesthat is different to a nucleic acid in its natural state (i.e. havingless than 100% sequence identity to a naturally occurring nucleic acidsequence), b) one or more non-naturally occurring nucleotide monomers(which may result in a non-natural backbone or sugar that is not G, A, Tor C) and/or c) may contain one or more other modifications (e.g., anadded label or other moiety) to the 5′-end, the 3′ end, and/or betweenthe 5′- and 3′-ends of the nucleic acid.

In the context of a preparation, the term “non-naturally occurring”refers to: a) a combination of components that are not combined bynature, e.g., because they are at different locations, in differentcells or different cell compartments; b) a combination of componentsthat have relative concentrations that are not found in nature; c) acombination that lacks something that is usually associated with one ofthe components in nature; d) a combination that is in a form that is notfound in nature, e.g., dried, freeze dried, crystalline, aqueous; and/ore) a combination that contains a component that is not found in nature.For example, a preparation may contain a “non-naturally occurring”buffering agent (e.g., Tris, HEPES, TAPS, MOPS, tricine or MES), adetergent, a dye, a reaction enhancer or inhibitor, an oxidizing agent,a reducing agent, a solvent or a preservative that is not found innature.

The term “template-switching” refers to a reverse transcription reactionin which the reverse transcriptase switches template from an RNAmolecule to a synthetic oligonucleotide (which usually contains two orthree Gs at its 3′ end, thereby copying the sequence of the syntheticoligonucleotide onto the end of the cDNA. Template switching isgenerally described in Matz et al., Nucl. Acids Res. 1999 27: 1558-1560and Wu et al., Nat Methods. 2014 11: 41-6. In template switching (and asillustrated in FIG. 1) a primer hybridizes to a RNA molecule. Thisprimer serves as a primer for a reverse transcriptase that copies theRNA molecule to form a complementary cDNA molecule. In copying the RNAmolecule, the reverse transcriptase commonly travels beyond the 5′ endof the mRNA to add non-template nucleotides to the 3′ end of the cDNA(typically Cs). Addition of an oligonucleotide that has ribonucleotidesor deoxyribonucleotides that are complementary to the non-templatenucleotides added onto the cDNA (e.g., a “template switching”oligonucleotide that typically has two or three Gs at its 3′ end), thereverse transcriptase will jump templates from the RNA template to theoligonucleotide template, thereby producing a cDNA molecule that has thecomplement of the template switching oligonucleotide at it's 3′ end.

The term “RNAseH activity” refers to an activity that hydrolyzes the RNAin RNA/DNA hybrid. Many reverse transcriptases have an RNAseH activitythat can be inactivated by truncation or by substitution.

The term “primer” refers to an oligonucleotide that is capable, uponforming a duplex with a polynucleotide template, of acting as a point ofinitiation of nucleic acid synthesis and being extended from its 3′ endalong the template so that an extended duplex is formed. The sequence ofnucleotides added during the extension process is determined by thesequence of the template polynucleotide. Primers are of a lengthcompatible with their use in synthesis of primer extension products, andcan are in the range of between 8 to 100 nucleotides in length, such as10 to 75, 15 to 60, 15 to 40, 18 to 30, 20 to 40, 21 to 50, 22 to 45, 25to 40, and so on, more typically in the range of between 18 to 40, 20 to35, 21 to 30 nucleotides long, and any length between the stated ranges.Primers are usually single-stranded. Primers have a 3′ hydroxyl.

The term “primer extension” as used herein refers to both to thesynthesis of DNA resulting from the polymerization of individualnucleoside triphosphates using a primer as a point of initiation, and tothe joining of additional oligonucleotides to the primer to extend theprimer. Primers can incorporate additional features which allow for thedetection or immobilization of the primer but do not alter the basicproperty of the primer, that of acting as a point of initiation of DNAsynthesis. For example, primers may contain an additional nucleic acidsequence at the 5′ end which does not hybridize to the target nucleicacid, but which facilitates cloning of the amplified product. The regionof the primer which is sufficiently complementary to the template tohybridize is referred to herein as the hybridizing region. The terms“target region” and “target nucleic acid” refers to a region orsubsequence of a nucleic acid which is to be reverse transcribed.

A polypeptide comprising at least 300 contiguous amino acids of SEQ IDNO:1 is provided. The first 23 amino acids can be removed from MMLVreverse transcriptase without altering the polymerase activity of thatenzyme (see, e.g., Gu et al., J. Mol. Biol. 305: 341-359, Najmudin etal, J. Mol. Biol. 296 613-632 and Das et al., Protein Sci. 2001 10:1936-1941). As such, some embodiments, the polypeptide may have atruncated N-terminus relative to SEQ ID NO:1. In some embodiments, thepolypeptide may comprises amino acid residues 24-335 of SEQ ID NO:1,e.g., the entire contiguous sequence of SEQ ID NO:1. The presentpolypeptide may contain 5, 6, or 7 or more amino acid substitutionsrelative to the corresponding sequence in the wild type MMLV reversetranscriptase.

The polypeptide may additionally comprise an amino acid sequence that isat least 90% (e.g., at least 95%, at least 98%, at least 99% or at least100%) identical to at least 286 contiguous amino acids (e.g., at least300 contiguous amino acids, at least 325 contiguous amino acids or atleast 336 contiguous amino acids) of SEQ ID NO:2, where SEQ ID NO:2 isthe sequence of the C-terminal part of an MMLV reverse transcriptase,shown below:

-   -   WGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDP        VAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLL        DTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQE        GQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIY        RRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDT        STLLIENSSPNSRLIN (SEQ ID NO:2)

This additional sequence may be positioned C-terminal to the at least300 contiguous amino acids of SEQ ID NO: 1. It is known that as many as62 residues can be removed from the C-terminus of the MMLV reversetranscriptase (see, e.g., U.S. Pat. No. 5,017,492) without significantlyaltering the polymerase activity. As such, in some embodiments, theadditional amino acid sequence may lack the C-terminal 3, 5, 10, 12, 15,30, 50 or 62 amino acids of SEQ ID NO:2.

The MMLV reverse transcriptase has been crystallized (see, e.g., Das etal., Structure. 2004 12: 819-29), the structure-functional relationshipsin MMLV reverse transcriptase have been studied (see, e.g., Cote et al.,Virus Res. 2008 134: 186-202, Georgiadis et al., Structure. 1995 3:879-92 and Crowther et al., Proteins 2004 57: 15-26) and many mutationsin MMLV reverse transcriptase are known (see, e.g., Yasukawa et al., J.Biotechnol. 2010 150: 299-306, Arezi et al Nucleic Acids Res. 2009 37:473-81 and Konishi et al., Biochem. Biophys. Res. Commun. 2014454:269-74, among many others). It is also known that one can truncatethe MMLV reverse transcriptase from either ends, and add exogenoussequences to either end (see, e.g., U.S. Pat. No. 5,017,492), withoutabolishing activity. As such, MMLV reverse transcriptase variants arewell known.

In some embodiments, the polypeptide may additionally comprises anexogenous domain and/or a purification tag (e.g., a His tag or the like)at either terminus. In some embodiments, the polypeptide may comprise asequence-specific DNA binding protein domain, which domain has beenshown to increase the processivity of other polymerases (see, e.g., US2016/0160193). In some embodiments the sequence-specific DNA bindingprotein domain may be a DNA binding protein domain listed in thefollowing table (as found in US 2016/0160193).

DNA-binding protein Tfx BD-51 gi|499321160 AbrB/MazE/MraZ-like BD-52gi|499321199 “Winged helix” DNA-binding domain BD-54 gi|499322061Ribbon-helix-helix protein, copG family BD-62 gi|499321149 lambdarepressor-like BD-63 gi|499322443 DNA-binding domains Resolvase-likeBD-67 gi|499322676 “Winged helix” DNA-binding domain BD-71 gi|499322676“Winged helix” DNA-binding domain BD-74 gi|499322255 “Winged helix”DNA-binding domain BD-75 gi|499322388 “Winged helix” DNA-binding domainBD-81 gi|499322131 “Winged helix” DNA-binding domain BD-82 gi|499321342“Winged helix” DNA-binding domain BD-85 gi|499321130 “Winged helix”DNA-binding domain BD-86 gi|499322705 “Winged helix” DNA-binding domainBD-88 gi|499320855 “Winged helix” DNA-binding domain BD-89 gi|499322250“Winged helix” DNA-binding domain BD-91 gi|499321633 “Winged helix”DNA-binding domain BD-92 gi|490170077 “Winged helix” DNA-binding domainBD-93 gi|499321272 “Winged helix” DNA-binding domain BD-94 gi|499320919“Winged helix” DNA-binding domain BD-97 gi|499320853 “Winged helix”DNA-binding domain BD-98 gi|499321734 “Winged helix” DNA-binding domainBD-100 gi|499322439 “Winged helix” DNA-binding domain BD-102gi|499322707 “Winged helix” DNA-binding domain BD-109 gi|499321112HCP-like BD-02 gi|351675391 Helix-turn-helix domain, rpiR family BD-03gi|500479591 Helix-turn-helix domain, rpiR family BD-04 gi|15643984Bacterial regulatory proteins, lacl family BD-07 gi|15643711 Bacterialregulatory proteins, lacl family BD-08 gi|15643974 Bacterial regulatoryproteins, lacl family BD-09 gi|15643956 Bacterial regulatory proteins,lacl family BD-11 gi|500480095 lambda repressor-like DNA-binding BD-12gi|15643421 domains “Winged helix” DNA-binding domain BD-14 gi|15644350“Winged helix” DNA-binding domain BD-16 gi|24159093 “Winged helix”DNA-binding domain BD-18 gi|15643139 “Winged helix” DNA-binding domainBD-23 gi|15642807 “Winged helix” DNA-binding domain BD-24 gi|15643159“Winged helix” DNA-binding domain BD-30 gi|15643333 “Winged helix”DNA-binding domain BD-32 gi|15643055 “Winged helix” DNA-binding domainBD-37 gi|15643827 “Winged helix” DNA-binding domain BD-43 gi|15643699Homeodomain-like BD-45 gi|15643788

In some embodiments, the polypeptide may have reverse transcriptaseactivity. In these embodiments, the polypeptide may or may not have anRNAseH activity in addition to the reverse transcriptase activity.Examples of MMLV reverse transcriptase that lack the RNAseH activity areknown (see, e.g., Kotewicz et al., Nucleic Acids Res. 1988 16: 265-77and Schultz et al., J. Virol. 1996 70: 8630-8).

A method for reverse transcribing an RNA template is also provided. Insome embodiments, this method may comprise: (a) combining a primer, anRNA template, a reverse transcriptase comprising: i. at least 300contiguous amino acids of SEQ ID NO:1, as described above, and ii. anamino acid sequence that is at least 90% identical to at least 286contiguous amino acids of SEQ ID NO:2 (as described above) that isC-terminal to the at least 300 contiguous amino acids of SEQ ID NO:1, aswell as any other components that may be necessary or desirable toperform a reverse transcription (e.g., salt, nucleotides, RNAseinhibitor, buffer, etc.), to produce a reaction mix; and (b) incubatingthe reaction mix to produce cDNA copied from the RNA template. The RNAtemplate may be any type of RNA template, e.g., total RNA, polyA⁺ RNA,capped RNA, enriched RNA etc., and the RNA can be from any source, e.g.,bacteria, mammals, an in vitro transcription reaction, etc., methods forthe making of which are known. The RNA template may contain RNAmolecules that are at least 1 kb in length, e.g., at least 2 kb, atleast 3 kb or at least 5 kb and, in some embodiments, at least some ofthe molecules in the RNA template may have a GC content of at least 50%,at least 60%, at least 70%, or at least 80%. The primer in the reactionmix may be any type of primer, e.g., an oligo-dT primer, a random primeror a gene-specific primer, for example, which primers are commonly usedto make cDNA. In some embodiments, the reaction mix may comprise atemplate switching oligonucleotide, as described above.

In some embodiments, the reaction mix may be incubated at temperaturethat is higher than 42° C., e.g., at a temperature in the range of 45°C. to 60° C. In some embodiments, the reaction mix may be incubated at atemperature in the range of 42° C. to 45° C., 48° C. to 51° C., 51° C.to 54° C., 54° C. to 57° C., 57° C. to 60° C. or 60° C. to 65° C. Insome embodiments, the population of cDNA molecules produced by themethod may be at least 20%, at least 40%, at least 60%, or at least 80%full length. The polymerase can reverse transcribe GC rich templatemolecules with increased efficiency compared with previously availablereverse transcriptases where the template molecules may have at least20%, 30%, 40%, 50%, 60%, 70% or 80% GC content (see for example, FIG.3).

If an oligo-dT or a random primer is used in the method, then the may beused to make a cDNA library that can be sequenced or used for geneexpression analysis. Alternatively, if a gene-specific primer is used,then method may be used for RT-PCR (e.g., quantitative RT-PCR) and othersimilar analyses.

All references cited herein are incorporated by reference.

EXAMPLES

Aspects of the present teachings can be further understood in light ofthe following examples, which should not be construed as limiting thescope of the present teachings in any way.

Superscript IV, Protoscript II and a variant MMLV reverse transcriptasereferred to as “M19H” were tested in a template switching assay todetermine the efficiency of reverse transcription of templates that havedifferent GC contents (14% to 88.6%). The M19H mutant comprises thefollowing N-terminal sequence:

(SEQ ID NO: 1) TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATATPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQELGDLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGIPTPKTPRQLREFLGTAGFCRLWIPGFAELAAPLYPLTKEGTLFN

A template switching assay was performed. 0.5 μl of 2 μM RNA transcript(200 to 330 nucleotides in length) of varying GC content (from 14% to88.6%), 0.3 μl of 1 μM FAM primer, 1 μl of 10 mM dNTP, 0.25 μl of RNaseInhibitor, 1 μl of 10 μM template switching oligonucleotide (oligo) and0.5 μl of reverse transcriptase were combined in a 10 μl reaction volume(using a buffer: 50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl₂, pH 8.3). Thereaction was incubated at a designated temperature (e.g., 50° C.) for 90minutes followed by inactivation step at 72° C. for 10 minutes. Afterthe reaction, incomplete products, full length products, and templateswitched products were quantified by a capillary electrophoresis assay(see Beckman Coulter (Indianapolis, Ind.) “Introduction to CapillaryElectrophoresis”). The areas under the peak of incompletion, elongationand template switching products were measured. The transcriptionefficiency equals to the sum of elongation and template switchingdivided by the sum of incompletion, elongation and template switching.The relationships between the components used in the assay are shown inFIG. 1. An example of how a capillary electrophoresis chromatogram canbe to quantify the incomplete products, full length products, andtemplate switched products is shown in FIG. 2.

Based on the results shown in FIG. 3, the M19H enzyme appears to be moreefficient than Superscript IV and Protoscript II, particularly for moreGC-rich templates, under the conditions used.

What is claimed is:
 1. A method comprising: (a) obtaining a reaction mixby combining a primer, an RNA template and a reverse transcriptase,wherein the reverse transcriptase comprises: at least 300 contiguousamino acids of SEQ ID NO: 1; and (b) incubating the reaction mix toproduce cDNA copied from the RNA template.
 2. The method of claim 1,wherein the reaction mix is incubated at temperature in the range of45−60° C.
 3. The method of claim 1, further comprises a templateswitching oligonucleotide.
 4. The method of claim 1, wherein the primeris an oligo-dT primer.
 5. The method of claim 1, wherein the primer is arandom primer.
 6. The method of claim 1, wherein the primer is agene-specific primer.
 7. The method of claim 1, wherein the reversetranscriptase comprises an exogenous sequence-specific DNA bindingdomain.
 8. The method of claim 1, wherein the reverse transcriptasecomprises an amino acid sequence that is at least 90% identical to atleast 300 contiguous amino acids of SEQ ID NO:2 that is C-terminal tothe at least 300 contiguous amino acids of SEQ ID NO:1.
 9. The method ofclaim 1, wherein the reverse transcriptase does not have RNAseHactivity.
 10. The method of claim 1, wherein the reverse transcriptasehas RNAseH activity.
 11. The method of claim 1, wherein the methodcomprises: (c) amplifying the cDNA produced in step (b).
 12. The methodaccording to claim 11, wherein the amplification is PCR.
 13. The methodof claim 1, wherein the method comprises quantifying the cDNA producedin step (b).
 14. A method comprising: (a) obtaining a reaction mix bycombining a primer, an RNA template and a reverse transcriptase, whereinthe reverse transcriptase comprises: (i) amino acids 24-335 of SEQ IDNO: 1 and (ii) an amino acid sequence that is at least 90% identical toamino acids 1-286 of SEQ ID NO: 2 wherein the amino acid sequence of (i)is N-terminal to the amino acid sequence of (ii); and (b) incubating thereaction mix to produce cDNA copied from the RNA template.